Abstract Radiotherapy, either alone, or in combination with chemotherapy, is frequently used in the treatment of patients with non-small cell lung carcinoma. However, tumor responses to ionizing radiation vary considerably and radiotherapy is not always effective in NSCLC patients. The epidermal growth factor receptor is an important determinant of radioresponse, whose elevated expression and activity frequently correlates with radioresistance in NSCLC tumors. We recently identified a subset of patient- derived NSCLC cell lines that exhibit marked sensitivity to radiation. A consistent feature among these NSCLCs is that they all harbor somatic, activating mutations in the tyrosine kinase domain (TKD) of the EGFR, that have also been linked to tumor sensitivity to small molecule tyrosine kinase inhibitors, gefitinib and erlotinib. We have assembled multiple lines of evidence that support a potential mechanism underlying mutant EGFR-associated radiosensitivity. The evidence reveals that, unlike the wild type EGFR, receptors with activating mutations in the TKD are not only defective in radiation- induced translocation to the nucleus but also fail to bind the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK). Consequently, NSCLCs with TKD-mutated EGFR exhibit significant delays in the repair of DNA double strand break repair and poor clonogenic survival in response to radiation. A crucial finding is that ectopic expression of TKD-mutated forms of EGFR can radiosensitize different cell lines with diverse genetic backgrounds, including radioprotective mutations in KRAS and p53. Our study has identified a subset of naturally occurring EGFR mutations that abrogate a critical radioprotective function of EGFR. We will use these as tools to address three specific aims of our proposal: (1) To confirm that NSCLCs with mutations in the tyrosine kinase domain of EGFR are defective in repair of radiation-induced DNA damage (2) To test the hypothesis that mutations in the tyrosine kinase domain of EGFR block radiation-induced nuclear translocation of EGFR and compromise DSB repair and clonogenic survival (3) To test the hypothesis that mutations in the tyrosine kinase domain of EGFR prevent radiation-induced EGFR-DNA-PK interactions and compromise DSB repair and clonogenic survival. The long term goal is to develop a molecular rationale for a radiotherapeutic strategy in NSCLC patients that simulates the radiosensitizing effects of TKD- mutated EGFR. Relevance Statement Nearly 75% of all lung cancers manifest as non-small lung carcinoma. The median five-year survival rate for patients diagnosed with non-small lung cancer (NSCLC) is estimated at barely 14%. Radiotherapy is the first and sometimes only line of treatment for patients with NSCLCs. However, tumor resistance to ionizing radiation presents a major challenge to radiotherapeutic control of NSCLCs. Efficient DNA repair pathways in radioresistant tumors are frequently augmented by molecular determinants of radioresponse such as the epidermal growth factor receptor (EGFR). One of the critical radioprotective functions of EGFR involves radiation induced nuclear transport and interactions with key enzymes in DNA repair pathways. The immediate objective is to elucidate mechanisms underlying the radiosensitivity associated with a biologically distinct class of NSCLCs that harbor mutations in the tyrosine kinase domain (TKD) of EGFR. The long term goal is to develop a radiotherapeutic strategy that simulates the radiosensitizing effects of TKD-mutated EGFR in NSCLC patients.